Radioactive Phospholipid Metal Chelates for Cancer Imaging and Therapy

ABSTRACT

R1 includes a chelating agent that is chelated to a metal atom, wherein the metal atom is a positron or single photon emitting metal isotope with a half life of greater than or equal to 4 hours, or an alpha, beta or Auger emitting metal isotope with a half life of greater than 6 hours and less than 30 days; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is —H, —OH, —COOH, —COOX, —OCOX, or —OX, wherein X is an alkyl or an arylalkyl; R2 is —N+H3, —N+H2Z, —N+HZ2, or —N+Z3, wherein each Z is independently an alkyl or an aroalkyl; and b is 1 or 2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/343,604filed Nov. 4, 2016, which claims the benefit of U.S. provisionalApplication No. 62/366,344 filed on Jul. 25, 2016. Each of theseapplications is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE DISCLOSURE

This disclosure relates generally to disease treatment and medicaldiagnosis/imaging. In particular, the disclosure is directed to thealkylphosphocholine analogs that include chelated radioactive metalisotopes to target and treat a wide range of pediatric and adultmalignant solid tumors, and to detect/image malignant solid tumor cells.

BACKGROUND

There are currently a variety of radiopharmaceuticals available fortumor imaging, but these are limited by non-specificity for malignancy,the inability to distinguish cancer from inflammation, short biologicalhalf-life, and generally poor spatial resolution associated with PET andSPECT scanning modalities.

We have previously shown that certain alkylphosphocholine analogs arepreferentially taken up and retained by malignant solid tumor (i.e.,solid tumor cancer) cells. In U.S. Patent Publication No. 2014/0030187,which is incorporated by reference herein in its entirety, Weichert etal. disclose using analogs of the base compound18-(p-iodophenyl)octadecyl phosphocholine (NM404; see FIG. 1) fordetecting and locating, as well as for treating, a wide variety of solidtumor cancers. For example, if the iodo moiety is an imaging-optimizedradionuclide, such as iodine-124 ([¹²⁴I]-NM404), the analog can be usedin positron emission tomography-computed tomography (PET/CT) orsingle-photon emission computed tomography (SPECT) imaging of adultsolid tumors. Alternatively, if the iodo moiety is a radionuclideoptimized for delivering therapeutic doses of radiation to the solidtumors cells in which the analog is taken up, such as iodine-125 oriodine-131 ([¹²⁵I]-NM404 or [¹³¹I]-NM404), the analog can be used totreat solid tumors.

There are a few recognized issues with using compounds that includeradioactive iodine isotopes for targeted radiation cancer therapy and/orimaging. For example, 1-124, suffers from poor positron output (onlyabout 24% of the emissions are positrons), and it suffers further from aconfounding gamma emission (600 KeV) which actually interferes withnormal 511 keV PET detection. Iodine-131, as a radiotherapy isotope,also contains other emissions at other energies, which add undesiredradiation dosimetry to neighboring normal tissues, including bonemarrow. The beta particle range of I-131 is also quite long, whichcontributes to off target toxicity.

Accordingly, there is a need in the art for improved cancer targetingagents for use in targeted cancer radiation therapy and/or imagingapplications.

BRIEF SUMMARY

The current disclosure provides new radioactive phospholipid metalchelates that can be used as improved cancer imaging and/or radiotherapyagents. A variety of positron- and gamma-emitting metals suitable forPET or SPECT imaging are available for chelation, as well as a varietyof α-, β-, and Auger-emitting metal nuclides for targeted radiotherapy.For either imaging or radiotherapy, radioactive metal isotopes with aminimum physical decay half-life of 6 h are necessary, due to thepharmacokinetic profile of the molecular carrier.

The radiactive phospholipid metal chelate compounds disclosed hereinutilize an alkyl-phospholipid carrier combined with one of a variety ofmetal chelators that is chelated to a radioactive metal isotope.Although radioiodinated versions have been shown to target cancer cellsin vivo, the disclosed metallic chelates are structurally quitedifferent. Specifically, they have a different charge, much larger size,and more lipophilic chemical properties. Despite these differences, thedisclosed chelates exhibit formulation properties that render themsuitable for injection and possess suitable in vivo stability, whileretaining tumor selectivity.

The disclosed metal chelates are preferentially taken up by malignantsolid tumor cells, as compared to non-tumor cells. Preferential uptakeof such compounds can be used in the therapeutic treatment of malignantsolid tumors, as well as in malignant solid tumor detection/imagingapplications. In therapeutic treatment, the alkylphosphocholinetargeting backbone includes a chelated radioactive metal isotope thatlocally delivers therapeutic dosages of radiation to the malignant solidtumor cells that preferentially take up the metal chelates. Indetection/imaging applications, the alkylphosphocholine targetingbackbone includes a chelated radioactive metal isotope suitable foremitting signals that can be used in detection/imaging.

Accordingly, the disclosure encompasses a family of radioactivephospholipid metal chelate compounds, compounds that can be used ascancer imaging agents and/or therapeutic agents for targeted cancerradiotherapy.

In a first aspect, the disclosure encompasses a compound having theformula:

or a salt thereof R₁ includes or is a chelating agent that is chelatedto a metal atom, wherein the metal atom is a positron or single photonemitting metal isotope with a half life of greater than or equal to 4hours, or an alpha, beta or Auger emitting metal isotope with a halflife of greater than 6 hours and less than 30 days; a is 0 or 1; n is aninteger from 12 to 30; m is 0 or 1; Y is-H, —OH, —COOH, —COOX, —OCOX, or—OX, wherein X is an alkyl or an arylalkyl; R₂ is —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂,or —N⁺Z₃, wherein each Z is independently an alkyl or an aroalkyl; and bis 1 or 2.

In some embodiments, the metal atom is a positron or single photonemitting metal isotope with a half life of greater than or equal to 4hours. Such isotopes are particularly suited for use in imagingapplications. Non-limiting examples of such isotopes include Ga-66,Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44, Ga-67, In-111,and Tc-99m.

In some embodiments, the metal atom is an alpha, beta or Auger emittingmetal isotope with a half life of greater than 6 hours and less than 30days. Such isotopes are particularly suited for use in targetedradiotherapy applications. Non-limiting examples of such isotopesinclude Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105,Ra-223, Ac-225, As-211, Pb-212, and Th-227.

In some embodiments, the chelating agent is1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of itsderivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one ofits derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) orone of its derivatives;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or oneof its derivatives; 1,4,7-triazacyclononane, 1-glutaricacid-4,7-diacetic acid (NODAGA) or one of its derivatives;1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA) or one of its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) orone of its derivatives;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A)or one of its derivatives; diethylene triamine pentaacetic acid (DTPA),its diester, or one of its derivatives; 2-cyclohexyl diethylene triaminepentaacetic acid (CHX-A″-DTPA) or one of its derivatives; deforoxamine(DFO) or one of its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) or one of itsderivatives; or DADA or one of its derivatives, wherein DADA has thestructure:

In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In otherembodiments, a is 0 (aliphatic alkyl chain).

In some embodiments, m is 1 (acylphospholipid series).

In some embodiments, n is an integer between 12 and 20.

In some embodiments, Y is —OCOX, —COOX or —OX. In some such embodiments,X is —CH₂CH₃ or —CH₃.

In some embodiments, m is 0 (alkylphospholipid series).

In some embodiments, b is 1.

In some embodiments, n is 18.

In some embodiments, R₂ is —N⁺Z₃. In some such embodiments, each Z isindependently —CH₂CH₃ or —CH₃. In some such embodiments, each Z is —CH₃.

Non-limiting examples of the chelating agent that can be chelated to themetal atom include:

Non-limiting examples of the disclosed cancer imaging and/or therapeuticagents include:

In each case, the exemplary compound is chelated to the metal atom.

In a second aspect, the disclosure encompasses a composition thatincludes one or of the compounds described above, and a pharmaceuticallyacceptable carrier.

In a third aspect, the disclosure encompasses one or more of thecompounds described above for use in imaging cancer or cancerous cells.

In a fourth aspect, the disclosure encompasses one or more of thecompounds described above for use in treating cancer.

In a fifth aspect, the disclosure encompasses one or more of thecompounds described above for use in treating cancer for use inmanufacturing a medicament for treating or imaging cancer.

In a sixth aspect, the disclosure encompasses a method for treating acancer in a subject. The method includes the step of administering to asubject having cancer an effective amount of one or more of thecompounds described above, wherein the metal atom is an alpha, beta orAuger emitting metal isotope with a half life of greater than 6 hoursand less than 30 days.

In some embodiments of the method, the chelated metal isotope is Lu-177,Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223, Ac-225,As-211, Pb-212, or Th-227.

In some embodiments, the compound is administered by parenteral,intranasal, sublingual, rectal, or transdermal delivery. In some suchembodiments, the compound is administered intravenously. In someembodiments, the compound is administered intratumoraly.

In some embodiments, the subject is a human.

In some embodiments, the cancer that is treated is an adult solid tumoror a pediatric solid tumor. Non-limiting examples of cancers that couldbe treated include melanoma, neuroblastoma, lung cancer, adrenal cancer,colon cancer, colorectal cancer, ovarian cancer, prostate cancer, livercancer, subcutaneous cancer, squamous cell cancer, intestinal cancer,retinoblastoma, cervical cancer, glioma, breast cancer, pancreaticcancer, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma,Wilms' tumor, and pediatric brain tumors.

In a seventh aspect, the disclosure encompasses a method for inhibitingthe proliferation or growth of malignant cells. The method includes thestep of contacting one or more malignant cells with an effective amountof one or more of the compounds described above, wherein the metal atomis an alpha, beta or Auger emitting metal isotope with a half life ofgreater than 6 hours and less than 30 days.

Non-limiting examples of metal isotopes that could be used includeLu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105, Ra-223,Ac-225, As-211, Pb-212, and Th-227.

In some embodiments, the method is performed in vivo, ex vivo, or invitro.

In some embodiments, the malignant cells are adult solid tumor cells orpediatric solid tumor cells. Non-limiting examples of such cells includemelanoma cells, neuroblastoma cells, lung cancer cells, adrenal cancercells, colon cancer cells, colorectal cancer cells, ovarian cancercells, prostate cancer cells, liver cancer cells, subcutaneous cancercells, squamous cell cancer cells, intestinal cancer cells,retinoblastoma cells, cervical cancer cells, glioma cells, breast cancercells, pancreatic cancer cells, Ewings sarcoma cells, rhabdomyosarcomacells, osteosarcoma cells, retinoblastoma cells, Wilms' tumor cells, andpediatric brain tumor cells.

In an eighth aspect, the disclosure encompasses a method for detectingor imaging one or more cancer cells in a biological sample. The methodincludes the steps of (a) contacting the biological sample with one ormore of the compounds described above, wherein the metal atom is apositron or single photon emitting metal isotope with a half life ofgreater than or equal to 4 hours, whereby the compound is differentiallytaken up by malignant solid tumor cells within the biological sample;and (b) identifying individual cells or regions within the biologicalsample that are emitting signals characteristic of the metal isotope.

Non-limiting examples of metal isotopes that could be used includeGa-66, Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44, Ga-67,In-111, and Tc-99m.

In some embodiments, the step of identifying individual cells or regionswithin the biological sample that are emitting signals characteristic ofthe metal isotope is performed by positron emission tomography (PET)imaging, single-photon emission computed tomography (SPECT) imaging, orgamma camera planar imaging.

In some embodiments, the biological sample is part or all of a subject.

In some embodiments, the biological sample is obtained from a subject.

In some embodiments, the subject is a human.

In some embodiments, the cancer cells are adult solid tumor cells orpediatric solid tumor cells. Non-limiting examples of such cells includemelanoma cells, neuroblastoma cells, lung cancer cells, adrenal cancercells, colon cancer cells, colorectal cancer cells, ovarian cancercells, prostate cancer cells, liver cancer cells, subcutaneous cancercells, squamous cell cancer cells, intestinal cancer cells,retinoblastoma cells, cervical cancer cells, glioma cells, breast cancercells, pancreatic cancer cells, Ewings sarcoma cells, rhabdomyosarcomacells, osteosarcoma cells, retinoblastoma cells, Wilms' tumor cells, andpediatric brain tumor cells.

In a ninth aspect, the disclosure encompasses a method of diagnosingcancer in a subject. The method includes one or more of theimaging/detection steps outlined above. In the method, the biologicalsample is obtained from, part of, or all of a subject. If cancer cellsare detected or imaged in the method steps, the subject is diagnosedwith cancer.

In some embodiments, the cancer that is diagnosed is an adult solidtumor or a pediatric solid tumor. Non-limiting examples of such cancerinclude melanoma, neuroblastoma, lung cancer, adrenal cancer, coloncancer, colorectal cancer, ovarian cancer, prostate cancer, livercancer, subcutaneous cancer, squamous cell cancer, intestinal cancer,retinoblastoma, cervical cancer, glioma, breast cancer, pancreaticcancer, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma,Wilms' tumor, and pediatric brain tumors.

In a tenth aspect, the disclosure encompasses a method of monitoring theefficacy of a cancer therapy in a human subject. The method includesperforming one or more of the imaging/detection steps outlined above attwo or more different times on the biological sample, wherein thebiological sample is obtained from, part of, or all of a subject. Thechange in strength of the signals characteristic of the metal isotopebetween the two or more different times is correlated with the efficacyof the cancer therapy.

In some embodiments, the cancer therapy being monitored is chemotherapyor radiotherapy.

In an eleventh aspect, the disclosure encompasses a method of treatingcancer in a subject. The method includes performing one or more of theimaging/detection steps outlined above, wherein the biological sample ispart of or all of a subject. The method also includes the step ofdirecting an external radiotherapy beam to the identified individualcells or regions within the subject.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of the base compound18-(p-iodophenyl)octadecyl phosphcholine (NM404).

FIG. 2 shows a time course MRI image of a tumor-bearing mouse followinginjection of Gd-DO3A-404 showing enhancement of the tumor (T) by 24hours.

FIG. 3 shows time course MRI images of tumor-bearing mice. The top panelincludes images of a mouse bearing a flank A549 (human NSCLC) tumorbefore contrast agent injection (left, arrow showing tumor location),one hour after injection of Gd-DO3A-404 (second from left), 24 hoursfollowing injection of Gd-DO3A-404 (third from left), and 48 hoursfollowing injection of Gd-DO3A-404 (rightmost image). The bottom panelincludes images of a mouse bearing a flank U87 (human glioma) tumorbefore contrast agent injection (leftmost image, arrow showing tumorlocation), one hour after injection of Gd-DO3A-404 (second from left),24 hours following injection of Gd-DO3A-404 (third from left), and 48hours following injection of Gd-DO3A-404 (rightmost image).

FIG. 4 shows further time course MRI images of tumor-bearing mice,continuing from FIG. 3. The top panel includes images of the mousebearing a flank A549 (human NSCLC) tumor three days after injection ofGd-DO3A-404 (leftmost image), four days following injection ofGd-DO3A-404 (second from left), and seven days following injection ofGd-DO3A-404 (rightmost image). The bottom panel includes images of themouse bearing a flank U87 (human glioma) tumor three days afterinjection of Gd-DO3A-404 (leftmost image), four days following injectionof Gd-DO3A-404 (second from left), and seven days following injection ofGd-DO3A-404 (rightmost image).

FIG. 5 is a bar graph of quantified results from the images shown inFIGS. 3 and 4. Specifically, the tumor to muscle T1-weighted signalratios are shown for both the mouse bearing a flank A549 (human NSCLC)tumor (shaded bar) and the mouse bearing a flank U87 (human glioma)tumor (unshaded bar) before contrast agent injection (pre), one hourafter injection of Gd-DO3A-404, 24 hours after injection of Gd-DO3A-404,48 hours after injection of Gd-DO3A-404, three days after injection ofGd-DO3A-404, four days after injection of Gd-DO3A-404, and seven daysafter injection of Gd-DO3A-404. *p<0.05 compared to pre-contrast, A549.^(#) p<0.05 compared to pre-contrast, U87.

FIG. 6 is a bar graph of quantified results from the images shown inFIGS. 3 and 4. Specifically, the tumor to muscle R₁ ratios are shown forboth the mouse bearing a flank A549 (human NSCLC) tumor (shaded bar) andthe mouse bearing a flank U87 (human glioma) tumor (unshaded bar) beforecontrast agent injection (pre-contrast), and 48 hours after injection ofGd-DO3A-404. *p<0.05 compared to pre-contrast, A549. ^(#) p<0.05compared to pre-contrast, U87.

FIGS. 7, 8, 9, 10, and 11 are T1-weighted spoiled gradient (SPGR)magnetic resonance (MR) images of three different mouse abdomencross-sections, showing in vivo biodistribution of the Gd-DO3A-404contrast agent.

FIG. 7 includes T1-weighted SPGR MR images obtained before the contrastagent is injected. The locations of the myocardium (M, top image), liver(L, center image), and kidney (K, bottom image) are indicated by arrows,and are consistent with the corresponding images shown in FIGS. 9-12.

FIG. 8 includes T1-weighted SPGR MR images obtained one hour afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 9 includes T1-weighted SPGR MR images obtained 24 hours afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 10 includes T1-weighted SPGR MR images obtained four days afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 11 includes T1-weighted SPGR MR images obtained seven days afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 12 shows a time course MRI image of tumor-bearing (U87) mice before(pre) and for various times following injection of DOTA-chelated Gd³⁺(DOTAREM®, top panel) and Gd-DO3A-404 (bottom panel). Tumor location inthe mouse flank is indicated by the arrow in the two “pre” images.

FIG. 13 is a bar graph is a bar graph of quantified results from theimages shown in FIG. 13. Specifically, the tumor to muscle signal ratiosare shown for both the U87 mouse before (pre) and at various times afterinjection with DOTAREM® (shaded bars) or Gd-DO3A-404 (unshaded bars).*p<0.05 compared to pre-contrast, DOTAREM®. ^(#) p<0.05 compared topre-contrast, Gd-DO3A-404.

FIG. 14 shows MRI brain images of orthotopic glioblastoma model mice.2.5 mg (top panel) or 3.7 mg (bottom panel) of Gd-DOA3A-404 wasadministered to the mice by intravenous injection, and these images wereobtained 48 hours after contrast agent injection.

FIG. 15 is a bar graph showing tissue biodistribution of Gd-DO3A-404 inxenograft A549-flank bearing mice 72 hours post-administration. n=3mice.

FIG. 16 shows time course MRI images obtained from a transgenic mousetriple-negative breast cancer model (n=4; Animals/rows 1-4). Alpha-betacrystalline overexpressing mice were imaged pre-administration (leftmostcolumn) and 24 hours (center column) and 48 hours (rightmost column)post-administration.

FIG. 17 shows T1-weighted images obtained from orthotopic xenograftmouse models. NOD-SCID mice with orthotopic U87 xenografts were imagedpre-administration, 24 hours, and 48 hours post administration ofGd-DO3A-404 (left panel). Orthotopic GSC 115 was imaged at 24 hours postadministration (right panel). GSC is a human glioma stem cell modelwhich was isolated from a human glioma patient.

FIG. 18 shows T1-weighted scans of a U87 flank xenograft bearing ratusing a clinical 3.0 T PET/MR. Rats were imaged pre- and 24 hourspost-administration of Gd-DO3A-404.

FIG. 19 shows simultaneous PET/MR images of a U87-flank bearing rat 24hours post-administration of Gd-DO3A-404 and Cu-DO3A-404. Gd-DO3A-404and 64Cu-DO3A-404 and were simultaneously administered to a U87-flankbearing rat. The rat was imaged using simultaneous PET/MR. Arrow pointsto tumor.

DETAILED DESCRIPTION I. In General

It is understood that this disclosure is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by any later-filednonprovisional applications.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising” andvariations thereof do not have a limiting meaning where these termsappear in the description and claims. Accordingly, the terms“comprising”, “including”, and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

The disclosure is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “effective amount,” as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include, without limitation,various lactose, mannitol, oils such as com oil, buffers such as PBS,saline, polyethylene glycol, glycerin, polypropylene glycol,dimethylsulfoxide, an amide such as dimethylacetamide, a protein such asalbumin, and a detergent such as Tween 80, mono- andoligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

The term “administering” or “administration,” as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

A route of administration in pharmacology is the path by which a drug istaken into the body. Routes of administration may be generallyclassified by the location at which the substance is applied. Commonexamples may include oral and intravenous administration. Routes canalso be classified based on where the target of action is. Action may betopical (local), enteral (system-wide effect, but delivered through thegastrointestinal tract), or parenteral (systemic action, but deliveredby routes other than the GI tract), via lung by inhalation.

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

Non-limiting examples for topical administrations may includeepicutaneous (application onto the skin), e.g., allergy testing ortypical local anesthesia, inhalational, e.g. asthma medications, enema,e.g., contrast media for imaging of the bowel, eye drops (onto theconjunctiva), e.g., antibiotics for conjunctivitis, ear drops, such asantibiotics and corticosteroids for otitis externa, and those throughmucous membranes in the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

Examples of parenteral administrations may include intravenous (into avein), e.g. many drugs, total parenteral nutrition intra-arterial (intoan artery), e.g., vasodilator drugs in the treatment of vasospasm andthrombolytic drugs for treatment of embolism, intraosseous infusion(into the bone marrow), intra-muscular, intracerebral (into the brainparenchyma), intracerebroventricular (into cerebral ventricular system),intrathecal (an injection into the spinal canal), and subcutaneous(under the skin). Among them, intraosseous infusion is, in effect, anindirect intravenous access because the bone marrow drains directly intothe venous system. Intraosseous infusion may be occasionally used fordrugs and fluids in emergency medicine and pediatrics when intravenousaccess is difficult.

As used herein, the term “intraperitoneal injection” or “IP injection”refers to the injection of a substance into the peritoneum (bodycavity). IP injection is more often applied to animals than to humans.In general, IP injection may be preferred when large amounts of bloodreplacement fluids are needed, or when low blood pressure or otherproblems prevent the use of a suitable blood vessel for intravenousinjection.

II. The Invention

In certain aspects, the disclosure is directed to alkylphosphocholineanalogs labeled with a radioactive metal isotope for detection/imagingof malignant tumor cells in a subject or in a biological sample. Thealkylphosphocholine analogs include a chelating moiety that chelates theradioactive metal isotope.

A. Radioactive Metal Isotopes for Malignant Solid Tumor Treatment

For the disclosed methods of therapeutically treating malignant solidtumors, any radioactive metal isotope known to emit ionizing radiationin a form that would result in the death of cells that take up theanalogs labeled with the radioactive metal isotope can be incorporatedby chelation into the alkylphosphocholine targeting backbone. In someembodiments, the radioactive metal isotope emits its ionizing radiationin a form that minimizes damage to tissue outside of the cells that takeup the labeled analogs.

Non-limiting examples of radioactive metal isotopes that could be usedinclude Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105,Ra-223, Ac-225, As-211, Pb-212, or Th-227.

B. Radioactive Metal Isotopes for Malignant Solid TumorDetection/Imaging

For the disclosed methods of detecting/imaging malignant solid tumors,any radioactive metal isotope known to emit radiation in a form that isreadily detectable by conventional imaging means can be incorporatedinto the targeting backbone. Non-limiting examples of “conventionalimaging means” include gamma ray detection, PET scanning, and SPECTscanning. Non-limiting examples of radioactive metal isotopes that couldbe used include Ga-66, Cu-64, Y-86, Co-55, Zr-89, Sr-83, Mn-52, As-72,Sc-44, Ga-67, In-111, or Tc-99m.

C. Metal Chelates of PLE Analogs

The disclosed structures utilize an alkylphosphocholine carrierbackbone. Once synthesized, the agents must harbor formulationproperties that render them suitable for injection while retaining tumorselectivity. A non-limiting exemplary series of metal chelate-PLEanalogs follows (additional non-limiting examples were describedpreviously). The structures shown include a chelating moiety to whichthe radioactive metal isotope is chelated to produce the final imagingor therapeutic agent.

D. Methods of Synthesizing Exemplary M-PLE Analogs

Proposed synthesis of compound 1 is shown below. The first step of thesynthesis is similar to described in Org Synth, 2008, 85, 10-14. Thesynthesis is started from cyclen which is converted into DO3A tris-Bnester. This intermediate is then conjugated with NM404 in the presenceof the base and Pd catalyst. Finally, benzyl protecting groups areremoved by the catalytic hydrogenation.

Synthesis of compound 2 is shown below. It begins with DO3A tris-Bnester which is alkylated with 3-(bromo-prop-1-ynyl)-trimethylsilane.After alkylation, the trimethjylsilyl group is removed and theintermediate acetylene is coupled with NM404 by the Sonogashirareaction. The benzyl groups are removed and the triple bond ishydrogenated simultaneously in the last step of the synthesis.

Compounds 5 and 6 can be synthesized from same precursors, DTPAdianhydride and 18-p-(3-hydroxyethyl-phenyl)-octadecyl phosphocholine asshown in the schemes below.

NOTA-NM404 conjugates can be synthesized in an analogous manner. Oneexample of NOTA-NM404 conjugate 7:

E. Dosage Forms and Administration Methods

Any route of administration may be suitable for administering thedisclosed alkylphosphocholine analogs to a subject. In one embodiment,the disclosed alkylphosphocholine analogs may be administered to thesubject via intravenous injection. In another embodiment, the disclosedalkylphosphocholine analogs may be administered to the subject via anyother suitable systemic deliveries, such as parenteral, intranasal,sublingual, rectal, or transdermal administrations.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via nasal systems or mouth through, e.g.,inhalation.

In another embodiment, the disclosed alkylphosphocholine analogs may beadministered to the subject via intraperitoneal injection or IPinjection.

In certain embodiments, the disclosed alkylphosphocholine analogs may beprovided as pharmaceutically acceptable salts. Other salts may, however,be useful in the preparation of the alkylphosphocholine analogs or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts include, without limitation, acid addition salts whichmay, for example, be formed by mixing a solution of thealkylphosphocholine analog with a solution of a pharmaceuticallyacceptable acid such as hydrochloric acid, sulphuric acid,methanesulphonic acid, fumaric acid, maleic acid, succinic acid, aceticacid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonicacid or phosphoric acid.

Where the disclosed alkylphosphocholine analogs have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thedisclosed alkylphosphocholine analogs possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present disclosure.

The disclosure also includes methods of using pharmaceuticalcompositions comprising one or more of the disclosed alkylphosphocholineanalogs in association with a pharmaceutically acceptable carrier.Preferably these compositions are in unit dosage forms such as tablets,pills, capsules, powders, granules, sterile parenteral solutions orsuspensions, metered aerosol or liquid sprays, drops, ampoules,auto-injector devices or suppositories; for parenteral, intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutically acceptable carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixturefor a compound of the present invention, or a pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe easily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid pre-formulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. Typical unit dosage forms contain from 1 to 100 mg,for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient.The tablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich, serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the alkylphosphocholine analogs may beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil or peanut oil, as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic and natural gums such as tragacanth,acacia, alginate, dextran, sodium caboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The disclosed alkylphosphocholine analogs are particularly useful whenformulated in the form of a pharmaceutical injectable dosage, includingin combination with an injectable carrier system. As used herein,injectable and infusion dosage forms (i.e., parenteral dosage forms)include, but are not limited to, liposomal injectables or a lipidbilayer vesicle having phospholipids that encapsulate an active drugsubstance. Injection includes a sterile preparation intended forparenteral use.

Five distinct classes of injections exist as defined by the USP:emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use. Powder forsuspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization. Solution injection involves a liquidpreparation containing one or more drug substances dissolved in asuitable solvent or mixture of mutually miscible solvents that issuitable for injection.

Solution concentrate injection involves a sterile preparation forparenteral use that, upon addition of suitable solvents, yields asolution conforming in all respects to the requirements for injections.Suspension injection involves a liquid preparation (suitable forinjection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. Examples Summary:

In Example 1, we provide an exemplary synthesis that also finds use forthe synthesis of analogous compounds chelating radioactive metalisotopes.

In Example 2, we demonstrate that an analog having a chelating agent andchelated metal substituted for the iodine moiety of NM404 (Gd-DO3A-404)is taken up by (and can be imaged in) solid tumor tissue, thus providingproof of concept for using the disclosed metal chelates as TRT and/orimaging agents.

In Example 3, we extended the results of Example 2 to demonstrate thetumor-targeting capabilities and uptake dynamics of Gd-DO3A-404 in twodifferent tumor models.

In Example 4, we report in-vivo biodistribution data for Gd-DO3A-404.

In Example 5, we demonstrate that the tumor-targeting properties ofGd-DO3A-404 reside in the NM404 targeting moiety. Specifically, wecompare the tumor uptake and retention data for Gd-DO3A-404 with thesame data obtained using DOTA-chelated Gd³⁺ (DOTAREM®).

In Example 6, we demonstrate Gd-DO3A-404 uptake in an orthotopicglioblastoma model.

In Example 7, we disclose biodistribution data for Gd-DOA-404 afterbeing administered to flank A549 xenograft mice.

In Example 8, we demonstrate Gd-DO3A-404 uptake in a triple-negativebreast cancer model.

In Example 9, we demonstrate Gd-DO3A-404 uptake in two orthotopicxenograft models.

In Example 10, we demonstrate simultaneous uptake and imaging (PET andMRI) of the gadolinium chelate Gd-DO3A-404, acting as an MRI contrastagent, and the copper radionuclide Cu-64 chelate 64Cu-DO3A-404, whichacts as a PET contrast agent.

Example 1: Synthesis of Metal Chelated DO3A-404

In this Example, we show the synthetic scheme used to synthesize oneexemplary phospholipid chelate, Gd-DO3A-404. Analogs incorporatingvarious radioactive isotopes could be synthesized in a similar manner,where the radioactive isotope in questions is substituted for Gd.

Scheme for synthesizing Gd-DO3A-404 (the disclosed radioactive metalisotopes can be readily substituted for Gd):

Example 2: In Vivo Imaging Proof of Concept

In this example, we demonstrate the successful in vivo MRI imaging of atumor, using Gd-DO3A-404 as the MRI contrast agent. The datademonstrates that the backbone phospholipid and chelating agent aretaken up and retained by solid tumors, demonstrating that such chelatesincorporating various radioactive metals, as disclosed herein, wouldexhibit similar properties

For proof-of-concept in vivo imaging of tumor uptake of the Gd-DO3A-404agent, nude athymic mouse with a flank A549 tumor (non small cell lungcancer) xenograft was scanned. The Gd-DO3A-404 agent (2.7 mg) wasdelivered via tail vein injection. Mice were anesthetized and scanningperformed prior to contrast administration and at 1, 4, 24, 48, and 72hours following contrast delivery. Imaging was performed on a 4.7 TVarian preclinical MRI scanner with a volume quadrature coil.T1-weighted images were acquired at all imaging time points using a fastspin echo scan with the following pulse sequence parameters: repetitiontime (TR)=206 ms, echo spacing=9 ms, echo train length=2, effective echotime (TE)=9 ms, 10 averages, with a 40×40 mm² field of view, 192×192matrix, 10 slices of thickness 1 mm each.

As seen in FIG. 2, MRI imaging of the tumor was significantly enhancedby 24 hours post-injection.

These results demonstrate that the differential uptake and retention ofalkylphosphocholine analogs is maintained for the metal chelated analogsdisclosed herein. Thus, the disclosed metal chelates can readily beapplied to clinical therapeutic and imaging applications.

Example 3: In Vivo Cancer Imaging in Multiple Tumor Models

In this extension of Example 2, we demonstrate selective uptake and invivo MRI imaging in two distinct flank tumor types, using Gd-DO3A-404 asthe MRI contrast agent.

To test uptake and retention in rodent models of human cancer, flankxenografts were established in mice for two distinct tumor types, A549(human non small cell lung cancer, NSCLC) and U87 (human glioma). N=3for each model. For pre-contrast imaging, T₁-W images of the tumor andabdomen (FIG. 4; 2 leftmost images) and T₁ maps of the tumor wereobtained.

At time zero (“contrast”), 2.5 mg of Gd-DO3A-404 (˜12 mmol/kg body mass)was delivered into the mice by intravenous injection. Animals werescanned pre-contrast and at various time points between one hour andseven days post-contrast (after one hour, 24 hours, 48 hours, threedays, four days and seven days). T₁ maps of the tumor were acquired foreach time point, along with T₁-weighted images of the tumor and theabdomen (see FIGS. 3 and 4).

In the NSCLC model, Gd-DO3A-404 uptake was not immediate and reached amaximum at 24-48 hours post-contrast (FIG. 3). The uptake was maintainedover several days (FIG. 4). In the U87 model, uptake was more rapid(already observable at one hour after delivery; see FIG. 3) and appearedto reach higher levels and was maintained for a longer time period (seeFIG. 4).

Those observations were confirmed by the quantified data, where tumor tomuscle T₁-weighted signal ratios were approximately doubled followingGd-DO3A-404 delivery (FIG. 5). The increase in tumor signal was morerapid and more prolonged in U87 tumors as compared to A549 tumors. Asseen in FIG. 6, the R₁ relaxation rate for both tumor types wassignificantly increased at 48 hours post-contrast.

In the disclosed metal chelates, a radioactive metal isotope is usedinstead of gadolinium. However, the rest of the structure, includingboth the tumor-targeting phospholipid moiety and the chelating agent,read on the disclosed metal chelates. Thus, the results demonstrate thatthe differential uptake and retention of alkylphosphocholine analogs inmultiple tumor types is maintained for the metal chelated analogsdisclosed herein. Accordingly, the disclosed metal chelates would beuseful in clinical cancer therapeutic and imaging applications.

Example 4: Use of MRI to Determine In Vivo Biodistribution ofGd-DO3A-404

In this Example, we determined the in vivo biodistribution of theGd-DO3A-404 after the contrast agent was administered (see Example 3).During the course of performing the experiments described in Example 4,we also acquired T₁-weighted spoiled gradient (SPGR) images in theabdomen of the mice, to observe biodistribution. Abdominalcross-sections imaged included the myocardium (FIGS. 7-11, top image),the liver (FIGS. 7-11, center image), and a kidney (FIGS. 7-11, bottomimage). Images are shown pre-contrast (FIG. 7), and at one hour (FIG.8), 24 hours (FIG. 9), four days (FIG. 10) and seven days post-contrast(FIG. 11).

In the myocardium and blood pool, the Gd-DO3A-404 contrast agentcirculates for nearly up to a day, after which any signal observed isdue to retention rather than from further uptake. In the liver andkidney, the Gd-DO3A-404 contrast agent is substantially cleared overtime, with more rapid clearance occurring through the liver, and moreprolonged clearance occurring through the kidney. Notably, theGd-DO3A-404 contrast agent exhibits a P-kinetic profile, includinghepatobiliary excretion, that is similar to that of relatedalkylphosphocholine analogs.

Example 5 The Gd-DO3A-404 Targeting Moiety Facilitates Tumor-Selectiveand Sustained Uptake

In this Example, we demonstrate that the selective uptake and retentionof Gd-DO3A-404 in tumor tissues is in fact facilitated by thetumor-targeting phospholipid moiety (the “404” moiety; see FIG. 1),rather than by the the gadolinium metal or its chelating agent.Accordingly, this Example demonstrates that effective tumor-targetingagents are not limited to those having a specific chelating agent ormetal ion, as long as they include the disclosed tumor-targetingphospholipid moieties.

To verify that uptake and retention was due to targeting of the “404”moiety, we directly compared the uptake of Gd-DO3A-404 with that ofDOTA-chelated Gd³⁺ (DOTAREM®) in an identical tumor model (mice withflank U87 tumors) and imaging scenario, using the same number of molesof each. As seen in FIG. 12, the uptake and clearance of DOTAREM®, ismuch more rapid than that of Gd-DO3A-404.

We the quantified the tumor to muscle ratio and compare it to baselinescans. As seen in FIG. 13, the DOTOREM® uptake was less striking, andsignificant only at a couple of early time points, as compared toGd-DO3A-404.

These results show that the phospholipid targeting moiety of Gd-DO3A-404(the 404 moiety), not the chelating agent and chelated metal (theGd-DO3A moiety) are responsible for the observed selective tumor uptakeand retention. Thus, a variety of different chelating moieties andchelated metals can be used without affecting the selective tumor uptakeand retention properties of the disclosed chelates.

Example 6: Brain Tumor Uptake of Gd-DO3A-404 in Orthotopic Glioma Model

In this Example, we demonstrate that at higher dosages, Gd-DO3A-404 canpass through the blood-brain barrier to successfully target brain tumortissue.

To investigate the use of Gd-DO3A-404 to detect tumors and metasteses insitu, in particular, in the brain, we created an orthotopic glioblastomamodel using cancer stem cells injected into the brain. To create themodel, brains of mice were injected with cells from orthotopicglioblastoma stem cell line 12.6. After sufficient tumor growth,monitored with T₂-weighted MRI, we imaged subjects pre-contrast andafter delivery (24-72 hours) of two different doses of Gd-DO3A-404 (2.5or 3.7 mg; ˜0.12-0.18 mmole/kg).

At the lower dose used for flank xenografts, no brain uptake wasobserved (see FIG. 14, upper panel). Because the lower delivered dosewas relatively low (on the same order of that delivered per kg bodyweight in clinical settings), we increased the dose for another group ofanimals. In this group, we observed uptake in one subject (FIG. 14,bottom panel). This result indicates that the blood-brain barrier (BBB)may be playing a role in brain tumor uptake, and dosage may be “tuned”to facilitate the contrast agent's passage through the BBB.

Example 7: In Vivo Biodistribution Data for Gd-DO3A-404 in Flank A549Xenograft Mice

In this extension of Example 4, we further examined the in vivobiodistribution of Gd-DO3A-404 after it is administered. Specifically,tissue biodistribution was measured in A549-flank bearing mice 72 hoursafter administration of Gd-DO3A-404. Nude athymic mice were sacrificed,perfused and tissues were collected and quantitated for Gd byhigh-resolution (magnetic-sector) inductively-coupled plasma massspectrometry (SF-ICPMS). n=3 mice.

As seen in FIG. 15, the Gd-DO3A-404 was selectively taken up by tumortissue, again demonstrating the suitability of the disclosedalkylphosphocholine analogs for targeted delivery to tumor tissue.

Example 8: Uptake of Gd-DO3A-404 in Triple-Negative Breast Cancer Model

In this example, we demonstrate the successful targeting of Gd-DO3A-404to breast cancer tissue.

Alpha-beta crystalline overexpressing mice (a triple negative breastcancer model) underwent MR imaging pre administration and 24 hours and48 hours post-administration of Gd-DO3A-400 (n=4). As seen in FIG. 16,over 48 hours, the contrast agent was taken up by and localized to thebreast cancer tissue.

This example illustrates that the disclosed alkylphosphocholine metalchelates can be used to target a wide range of solid tumor tissues.

Example 9: Uptake of Gd-DO3A-404 in Orthotopic Model

In this example, we demonstrate the successful targeting of Gd-DO3A-404in two different orthotopic xenograft models.

NOD-SCID mice with orthotopic U87 xenografts were imagedpre-administration, 24 hours, and 48 hours post administration ofGd-DO3A-404. As seen in FIG. 17 (left panel), the contrast agent wasdifferentially taken up by the tumor tissue (see arrows).

Orthotopic GSC 115 was imaged at 24 hours post administration ofGd-DO3A-404. GSC is a human glioma stem cell model which was isolatedfrom a human glioma patient. As seen in FIG. 17 (right panel), thecontrast agent was differentially taken up by the tumor tumor tissue(see arrow).

This example illustrates that the disclosed alkylphosphocholine metalchelates can be used to target a wide range of solid tumor tissues.

Example 10: Simultaneous PET/MR Imaging Demonstrating Tumor Targeting byBoth Gd-DO3A-404 and ⁶⁴Cu-DO3A-404

In this example, we demonstrate the successful use of both Gd-DO3A-404and ⁶⁴Cu-DO3A-404 as tumor targeting contrast agents (Gd-DO3A-404 forMRI and ⁶⁴Cu-DO3A-404 for simultaneous PET imaging).

Simultaneous imaging was performed using a clinical Pet/MRI scanner.⁶⁴Cu-DO3A-404 has the same structure as Gd-DO3A-404, except that ⁶⁴Cu, apositron emitting radionuclide, is chelated to the chelating moietyinstead of Gd. ⁶⁴Cu-DO3A-404 was synthesized (and can be synthesizedusing the methods disclosed herein; see, e.g., Example 1). Both the⁶⁴Cu-DO3A-404 and Gd-DO3A-404 chelates were injected simultaneously intoa rat with a flank U87 (human glioma) xenograft.

T1-weighted scans of the U87 flank xenograft were obtained using theclinical 3.0 T PET/MR. Rats were imaged pre- and 24 hourspost-administration of the Gd-DO3A-404. The resulting MR imagesdemonstrate selective tumor uptake of the Gd-DO3A-404 contrast agent(FIG. 18; arrow showing tumor location).

Simultaneous PET/MR scans of the U87-flank bearing rat 24 hourspost-simultaneous administration of both Gd-DO3A-404 (the MRI contrastagent) and ⁶⁴Cu-DO3A-404 (the PET contrast agent) were obtained. As seenin FIG. 19, fused T1-weighted MR and PET images showed excellentcolocalization of contrast and activity in the flank and abdomen (arrowpoints to tumor). The tumor is enhanced enhances in both the T1 and T2MRI images (FIG. 19). Furthermore, the simultaneous PET scandemonstrates tumor uptake of the ⁶⁴Cu-DO3A-404 PET contrast agent (FIG.19), providing proof-of concept for using the disclosed chelates havinga radioactive metal substituted for Gd in tumor imaging (such as PETimaging) and radiotherapy applications.

In sum, these examples demonstrate that radioactive metal chelates thatinclude an appropriate tumor-targeting phospholipid moiety, as disclosedherein, would demonstrate selective uptake and retention in multiplecancer types. Such chelates will facilitate improved detection,characterization, and staging of cancer and metastases. In addition,such agents can be used for radiotherapy targeted to the solid tumors inwhich they are selectively taken up and retained.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

1. A method for treating a cancer in a subject, comprising administeringto a subject having cancer an effective amount of a compound having theformula:

or a salt thereof, wherein: R₁ comprises a chelating agent that ischelated to a metal atom, wherein the metal atom is an alpha, beta orAuger emitting metal isotope with a half life of greater than 6 hoursand less than 30 days; a is 0 or 1; n is an integer from 12 to 30; m is0 or 1; Y is selected from the group consisting of —H, —OH, —COOH,—COOX, —OCOX, and —OX, wherein X is an alkyl or an arylalkyl; R₂ isselected from the group consisting of —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, and —N⁺Z₃,wherein each Z is independently an alkyl or an aroalkyl; and b is 1 or2; whereby the cancer is successfully treated in the subject.
 2. Themethod of claim 1, wherein the metal isotope is selected from the groupconsisting of Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199,Rh-105, Ra-223, Ac-225, As-211, Pb-212, and Th-227.
 3. The method ofclaim 1, wherein the chelating agent is selected from the groupconsisting of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A)and its derivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA)and its derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)and its derivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and its derivatives; 1,4,7-triazacyclononane, 1-glutaricacid-4,7-diacetic acid (NODAGA) and its derivatives;1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA) and its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) andits derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid (CB-TE2A) and its derivatives; diethylene triamine pentaacetic acid(DTPA), its diester, and its derivatives; 2-cyclohexyl diethylenetriamine pentaacetic acid (CHX-A″-DTPA) and its derivatives;deforoxamine (DFO) and its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) and itsderivatives; and DADA and its derivatives.
 4. The method of claim 1,wherein the chelating agent chelated to the metal atom is selected fromthe group consisting of:


5. The method of claim 5, wherein the compound is selected from thegroup consisting of:

wherein the selected compound is chelated to the metal atom.
 6. Themethod of claim 1, wherein the subject is a human.
 7. The method ofclaim 1, wherein the cancer that is treated is an adult solid tumor or apediatric solid tumor.
 8. The method of claim 7, wherein the cancer isselected from the group consisting of melanoma, neuroblastoma, lungcancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer,prostate cancer, liver cancer, subcutaneous cancer, squamous cellcancer, intestinal cancer, retinoblastoma, cervical cancer, glioma,breast cancer, pancreatic cancer, Ewings sarcoma, rhabdomyosarcoma,osteosarcoma, retinoblastoma, Wilms' tumor, and pediatric brain tumors.9. A method for detecting or imaging one or more cancer cells in abiological sample, comprising: (a) contacting the biological sample witha compound having the formula:

or a salt thereof, wherein: R₁ comprises a chelating agent that ischelated to a metal atom, wherein the metal atom is a positron or singlephoton emitting metal isotope with a half life of greater than or equalto 4 hours; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Yis selected from the group consisting of —H, —OH, —COOH, —COOX, —OCOX,and —OX, wherein X is an alkyl or an arylalkyl; R₂ is selected from thegroup consisting of —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, and —N⁺Z₃, wherein each Z isindependently an alkyl or an aroalkyl; and b is 1 or 2; whereby thecompound is differentially taken up by malignant solid tumor cellswithin the biological sample; and (b) identifying individual cells orregions within the biological sample that are emitting signalscharacteristic of the metal isotope, whereby one or more cancer cellsare detected or imaged.
 10. The method of claim 9, wherein the metalisotope is selected from the group consisting of Ga-66, Cu-64, Y-86,Co-55, Zr-89, Sr-83, Mn-52, As-72, Sc-44, Ga-67, In-111, and Tc-99m. 11.The method of claim 9, wherein the step of identifying individual cellsor regions within the biological sample that are emitting signalscharacteristic of the metal isotope is performed by positron emissiontomography (PET) imaging, single-photon emission computed tomography(SPECT) imaging, or gamma camera planar imaging.
 12. The method of claim9, wherein the chelating agent is selected from the group consisting of1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and itsderivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) and itsderivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and itsderivatives; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) and its derivatives; 1,4,7-triazacyclononane, 1-glutaricacid-4,7-diacetic acid (NODAGA) and its derivatives;1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA) and its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) andits derivatives; 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diaceticacid (CB-TE2A) and its derivatives; diethylene triamine pentaacetic acid(DTPA), its diester, and its derivatives; 2-cyclohexyl diethylenetriamine pentaacetic acid (CHX-A″-DTPA) and its derivatives;deforoxamine (DFO) and its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) and itsderivatives; and DADA and its derivatives.
 13. The method of claim 9,wherein the chelating agent chelated to the metal atom is selected fromthe group consisting of:


14. The method of claim 9, wherein the compound is selected from thegroup consisting of:

wherein the selected compound is chelated to the metal atom.
 15. Themethod of claim 9, wherein the biological sample is part or all of asubject.
 16. The method of claim 15, wherein the subject is a human. 17.The method of claim 9, wherein the cancer cells are adult solid tumorcells or pediatric solid tumor cells.
 18. The method of claim 17,wherein the cancer cells are selected from the group consisting ofmelanoma cells, neuroblastoma cells, lung cancer cells, adrenal cancercells, colon cancer cells, colorectal cancer cells, ovarian cancercells, prostate cancer cells, liver cancer cells, subcutaneous cancercells, squamous cell cancer cells, intestinal cancer cells,retinoblastoma cells, cervical cancer cells, glioma cells, breast cancercells, pancreatic cancer cells, Ewings sarcoma cells, rhabdomyosarcomacells, osteosarcoma cells, retinoblastoma cells, Wilms' tumor cells, andpediatric brain tumor cells.
 19. A method of diagnosing cancer in asubject, comprising performing the method of claim 9, wherein thebiological sample is obtained from, part of, or all of a subject, andwhereby if cancer cells are detected or imaged, the subject is diagnosedwith cancer.
 20. A method of treating cancer in a subject, comprisingperforming the method of claim 9, wherein the biological sample is partof or all of a subject, and directing an external radiotherapy beam tothe identified individual cells or regions within the subject.